1. Field of the Invention
The present invention relates to pharmaceutical preparations containing a human growth hormone having a molecular weight of about 20,000 (hereinafter referred to as 20 k hGH), more specifically to lyophilized preparations prepared from a solution containing 20 k hGH, which have excellent storage stability and do not produce any foreign or insoluble matter when reconstituted, and to processes for the production thereof.
2. Description of the Related Art
There are two known types of human growth hormone: one having a molecular weight of about 22,000 (22 k hGH) and the other having a molecular weight of about 20,000 (20 k hGH). The 22 k hGH is produced by means of recombinant DNA technology and is used for treatment of pituitary dwarfism in the field of pediatrics. The 20 k hGH has never been produced on an industrial scale and it has never been used for medical treatment.
20 k hGH is a single-chain polypeptide having a molecular weight of about 20,000 and has an isoelectric point of about 5.5. Thus, it is stable in an aqueous solution at a neutral pH but unstable at acid and alkaline pH ranges. The solubility of 20 k hGH in aqueous solutions at weak acid to weak alkaline pH ranges is less than about 1 mg/ml and insoluble matter is produced upon thawing out the frozen solution. Thus, 20 k hGH can be considered to be a protein with very low solubility. Furthermore, in aqueous solutions at weak acid to weak alkaline pH ranges, 20 k hGH easily dimerizes. It has been reported that 20 k hGH derived from the human pituitary gland often co-dimerizes or dimerizes with 22 k hGH, a human growth hormone having a molecular weight of about 22,000 (Chapman et al., J. Biol. Chem., Vol. 256, 2395-2401, 1981). These facts suggest that the low solubility of 20 k hGH is due to a hydrophobic interaction of protein molecules.
In order to improve the solubility of highly hydrophobic proteins, sodium dodecyl sulfate, which is extremely surface active, or denaturing agents such as urea and guanidine hydrochloride, and the like are generally used. However, these agents destroy the protein structure, and the primary functions of the proteins will be lost or weakened. Therefore, the use of these conventional agents are not at all preferable if the proteins are to be used in pharmaceutical preparations.
On the other hand, in one known example of the use of solubility promoters to improve the solubility of proteins, equimolar quantities of histidine and creatinine, having a positive charge, and citric acid, having a negative charge, were added to improve the solubility of a modified form of tissue plasminogen activator (hereinafter referred to as tPA) (U.S. Pat. No. 4,980,165). tPA and modified tPA are proteins which are extremely insoluble at neutral pH ranges but highly soluble at acid pH ranges. In other words, the low solubility of tPA and modified tPA is caused by isoelectric precipitation of the proteins, which this method suppresses by the addition of histidine and creatinine, having a positive charge, and citric acid, having a negative charge.
Prescriptions of pharmaceutical preparations containing a human growth hormone having a molecular weight of about 22,000, which are commercially available today, are shown in Table 1. These preparations are generally administered subcutaneously or intramuscularly.
Further, these 22 k hGH preparations primarily contain glycine or mannitol and are stable when stored at 5.degree. C. for 1 year.
TABLE 1 ______________________________________ Prescription of commercially available lyophilized 22k hGH Solution Name of product Additive(s) reconstituti on ______________________________________ Genotropin (Kabi 4IU Glycine: 24 mg Water for 1 ml Pharmacia Sumitomo) injection Norditropin (Nordisk) 4IU Glycine: 24 mg Water for 1 ml D-Mannitol: 2.4 mg injection Humatrope (Liliy) 4IU Glycine: 1.48 mg Saline 2 ml D-Mannitol: 7.4 mg Saizen (Serono) 4IU D-Mannitol: 20 mg Saline 1 ml Groject (Bio-Tech 4IU D-Mannitol: 40 mg Saline 1 ml General) ______________________________________
A study by the present inventors showed that stable preparations could not be obtained when lyophilized 20 k hGH preparations were produced as above.
If a stable aqueous solution of 20 k hGH of sufficient concentration cannot be obtained for use in producing preparations to be administered as described above, then the dosage would have to be increased. This can be extremely inconvenient in the case of 20 k hGH preparations for injection.
Several compounding methods are known to stabilize 22 k human growth hormones in solution. Reported examples include the addition of arginine and EDTA as stabilizing agents for an aqueous 22 k hGH solution (EP Publication No. 639984), the addition of polyhydric alcohols or amino acids in order to control the production of insoluble matter and maintain activity of soluble matter in a 22 k hGH solution (EP 303746), and the addition of histidine as a stabilizing agent in order to suppress an increase in related substances in an aqueous 22 k hGH solution (EP Publication No. 618807). However, all of these methods were developed to stabilize 22 k hGH in a solution, and do not refer to the stabilization of a lyophilized 22 k hGH product. Furthermore, nothing is known about stabilization of a lyophilized preparation of 20 k hGH.
The present inventors studied the solubility and stability of 20 k hGH. As shown in Table 2, the result showed that aside from being of lower molecular weight than 22 k hGH, 20 k hGH is quite different from 22 k hGH in physicochemical properties such as physiological activity, stability and solubility. In particular, the original 20 k hGH bulk solution is unstable even after lyophilization. When its lyophilized preparation is stored at a temperature as low as 5.degree. C., the quantities of related substances, such as a deamidated variant in which Asp.sup.134 in 20 k hGH is deamidated to Asp.sup.134 and a sulfoxide variant in which Met.sup.14 was converted to ox-Met.sup.4, and high molecular weight polymer products, increased over time. The related substances means, for example, a mono-deamidated variant in which Asn.sup.134 in 20 k hGH is deamidated to Asp.sup.134, a di-deamidated variant in which besides Asn.sup.134 , Asn.sup.137 was also deamidated, and a sulfoxide variant in which Met.sup.14 was converted to ox-Met.sup.14. Furthermore, in handling, for example, pipetting, a solution in which the 20 k hGH was dissolved, the protein were readily aggregated to produce insoluble matter. In other words, the stability in an aqueous solution is low. As described hereinafter, even the addition of basic amino acids to a 20 k hGH solution does not suppress the production of insoluble matter or related substances. As discussed above, 20 k hGH is highly hydrophobic, which may explain why it tends to produce more insoluble matter than 22 k hGH.
TABLE 2 ______________________________________ Difference in physicochemical properties between 20k hGH and 22k hGH Physicochemical property 20k hGH 22k hGH ______________________________________ Isoelectric point pH 5.5.sup.1 pH 5.1.sup.1 Dimer formation Easily formed.sup.2 Hardly formed.sup.2 Hydrophobicity High.sup.3 Low.sup.3 Solubility in water Low.sup.3 High.sup.3 Stability of lyophilized hGH Low.sup.3 High.sup.3 Stability of dissolved hGH Low.sup.3 High.sup.3 ______________________________________ .sup.1 : Endocrine Reviews, Vol. 12, 314-324, 1991. .sup.2 : J. Biol. Chem., Vol. 256, 2395-2401, 1981. .sup.3 : Data by the present inventors.